Combined mutations of ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, TET2 and WT1 genes in myelodysplastic syndromes and acute myeloid leukemias

<p>Abstract</p> <p>Background</p> <p>Gene mutation is an important mechanism of myeloid leukemogenesis. However, the number and combination of gene mutated in myeloid malignancies is still a matter of investigation.</p> <p>Methods</p> <p>We searched for mutations in the <it>ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, TET2 </it>and <it>WT1 </it>genes in 65 myelodysplastic syndromes (MDSs) and 64 acute myeloid leukemias (AMLs) without balanced translocation or complex karyotype.</p> <p>Results</p> <p>Mutations in <it>ASXL1 </it>and <it>CBL </it>were frequent in refractory anemia with excess of blasts. Mutations in <it>TET2 </it>occurred with similar frequency in MDSs and AMLs and associated equally with either <it>ASXL1 </it>or <it>NPM1 </it>mutations. Mutations of <it>RUNX1 </it>were mutually exclusive with <it>TET2 </it>and combined with <it>ASXL1 </it>but not with <it>NPM1</it>. Mutations in <it>FLT3 (</it>mutation and internal tandem duplication), <it>IDH1</it>, <it>IDH2</it>, <it>NPM1 </it>and <it>WT1 </it>occurred primarily in AMLs.</p> <p>Conclusion</p> <p>Only 14% MDSs but half AMLs had at least two mutations in the genes studied. Based on the observed combinations and exclusions we classified the 12 genes into four classes and propose a highly speculative model that at least a mutation in one of each class is necessary for developing AML with simple or normal karyotype.</p

Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice

International audienceAbstract Bistable motoneurons of the spinal cord exhibit warmth-activated plateau potential driven by Na + and triggered by a brief excitation. The thermoregulating molecular mechanisms of bistability and their role in motor functions remain unknown. Here, we identify thermosensitive Na + -permeable Trpm5 channels as the main molecular players for bistability in mouse motoneurons. Pharmacological, genetic or computational inhibition of Trpm5 occlude bistable-related properties (slow afterdepolarization, windup, plateau potentials) and reduce spinal locomotor outputs while central pattern generators for locomotion operate normally. At cellular level, Trpm5 is activated by a ryanodine-mediated Ca 2+ release and turned off by Ca 2+ reuptake through the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) pump. Mice in which Trpm5 is genetically silenced in most lumbar motoneurons develop hindlimb paresis and show difficulties in executing high-demanding locomotor tasks. Overall, by encoding bistability in motoneurons, Trpm5 appears indispensable for producing a postural tone in hindlimbs and amplifying the locomotor output

The M-current works in tandem with the persistent sodium current to set the speed of locomotion

International audienceThe central pattern generator (CPG) for locomotion is a set of pacemaker neurons endowed with inherent bursting driven by the persistent sodium current (INaP). How they proceed to regulate the locomotor rhythm remained unknown. Here, in neonatal rodents, we identified a persistent potassium current critical in regulating pacemakers and locomotion speed. This current recapitulates features of the M-current (IM): a subthreshold noninactivating outward current blocked by 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE991) and enhanced by N-(2-chloro-5-pyrimidinyl)-3,4-difluorobenzamide (ICA73). Immunostaining and mutant mice highlight an important role of Kv7.2-containing channels in mediating IM. Pharmacological modulation of IM regulates the emergence and the frequency regime of both pacemaker and CPG activities and controls the speed of locomotion. Computational models captured these results and showed how an interplay between IM and INaP endows the locomotor CPG with rhythmogenic properties. Overall, this study provides fundamental insights into how IM and INaP work in tandem to set the speed of locomotion

Antimicrobial capacity of the freshwater planarians against S-aureus is under the control of Timeless

International audiencePlanarians, which are non-parasitic flatworms, are highly resistant to bacterial infections. To better understand the mechanisms underlying this resistance, we investigated the role of the circadian machinery in the anti-bacterial response of the freshwater planarian Schmidtea mediterranea. We identified Smed-Tim from S. mediterranea as a homolog of the mammalian clock gene Tim. We showed via RNA interference that Smed-Tim is required for the antimicrobial activities of Schmidtea mediterranea against Staphylococcus aureus infection during the light/dark cycle. Indeed, S. aureus infection leads to the expression of Smed-Tim, which in turn promotes Smed-Traf6 and Smed-morn2, but not Smed-p38 MAPK expression, 2 master regulators of planarian anti-microbial responses

Knockdown of calpain1 in lumbar motoneurons reduces spasticity after spinal cord injury in adult rats

International audienceSpasticity, affecting $75% of patients with spinal cord injury (SCI), leads to hyperreflexia, muscle spasms, and cocontractions of antagonist muscles, greatly affecting their quality of life. Spasticity primarily stems from the hyperexcitability of motoneurons below the lesion, driven by an upregulation of the persistent sodium current and a downregulation of chloride extrusion. This imbalance results from the post-SCI activation of calpain1, which cleaves Nav1.6 channels and KCC2 cotransporters. Our study was focused on mitigating spasticity by specifically targeting calpain1 in spinal motoneurons. We successfully transduced lumbar motoneurons in adult rats with SCI using intrathecal administration of adeno-associated virus vector serotype 6, carrying a shRNA sequence against calpain1. This approach significantly reduced calpain1 expression in transduced motoneurons, leading to a noticeable decrease in spasticity symptoms, including hyperreflexia, muscle spasms, and cocontractions in hindlimb muscles, which are particularly evident in the second month post-SCI. In addition, this decrease, which prevented the escalation of spasticity to a severe grade, paralleled the restoration of KCC2 levels in transduced motoneurons, suggesting a reduced proteolytic activity of calpain1. These findings demonstrate that inhibiting calpain1 in motoneurons is a promising strategy for alleviating spasticity in SCI patients

Functional Proteomics Mapping of a Human Signaling Pathway

Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor β (TGFβ) superfamily. We used two-hybrid screening to map Smad signaling protein–protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach

<i>Tropheryma whipplei</i>, the Agent of Whipple's Disease, Affects the Early to Late Phagosome Transition and Survives in a Rab5- and Rab7-Positive Compartment

<div><p><i>Tropheryma whipplei</i>, the agent of Whipple's disease, inhibits phago-lysosome biogenesis to create a suitable niche for its survival and replication in macrophages. To understand the mechanism by which it subverts phagosome maturation, we used biochemical and cell biological approaches to purify and characterise the intracellular compartment where <i>Tropheryma whipplei</i> resides using mouse bone-marrow-derived macrophages. We showed that in addition to Lamp-1, the <i>Tropheryma whipplei</i> phagosome is positive for Rab5 and Rab7, two GTPases required for the early to late phagosome transition. Unlike other pathogens, inhibition of PI(3)P production was not the mechanism for Rab5 stabilisation at the phagosome. Overexpression of the inactive, GDP-bound form of Rab5 bypassed the pathogen-induced blockade of phago-lysosome biogenesis. This suggests that <i>Tropheryma whipplei</i> blocks the switch from Rab5 to Rab7 by acting on the Rab5 GTPase cycle. A bio-informatic analysis of the <i>Tropheryma whipplei</i> genome revealed a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologous with the GAPDH of <i>Listeria monocytogenes</i>, and this may be the bacterial protein responsible for blocking Rab5 activity. To our knowledge, <i>Tropheryma whipplei</i> is the first pathogen described to induce a “chimeric” phagosome stably expressing both Rab5 and Rab7, suggesting a novel and specific mechanism for subverting phagosome maturation.</p></div

<i>T. whipplei</i> survives and replicates in a Lamp-1- but not cathepsin D-positive compartment within BMDMs.

<p>(<b>A</b>) BMDMs were incubated with <i>T. whipplei</i> (bacterium-to-cell ratio of 50∶1) for 4 hours (day 0). Cells were then washed to remove free bacteria and incubated for additional periods (days). The copy number of bacterial DNA was determined by qRT-PCR. The results are expressed as the mean ± SD from 3 experiments (*<i>p</i><0.05). (<b>B–D</b>) The colocalisation of <i>T whipplei</i> with either (<b>B</b>) Lamp-1 or (<b>C</b>) cathepsin D was analysed in BMDMs by immunofluorescence and confocal microscopy. The percentage of <i>T whipplei</i> colocalising with either Lamp-1 or cathepsin D was quantified over the time (<b>D</b>). The results are expressed as the mean ± SD from 3 experiments (*<i>p</i><0.05). The scale bars indicate 5 µm.</p